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Adım: manyetik zenginleştirme ile dalak B hücreleri izolasyonu <ol><li> Deneysel farelerin bağışıklık sisteminin olgunlaşmasını sağlamak için 8-12 hafta arası olmalıdır. </li><li> Servikal dislokasyon fare Euthanize ve% 70 etanol ile ıslatın. Cerrahi karnın sol hypochondrium kas ve deri yoluyla bir kesi yaparak, dalak kaldırmak ve fosfat üç büyük parçalar halinde kesip tamponlu salin (PBS). Tüm alet ve reaktifler steril olduğundan emin olun. </li><li> Lapa hafifçe PBS içine 70 mikron hücre süzgecinden 1 ml şırınga pistonu kauçuk sonu dalak kullanarak. Taşlama büyük (yani prolifere) hücreleri rüptürüne yol açabilir gibi bir itme hareketi yerine taşlama hareket kullandığınızdan emin olun. </li><li> 5 dakika için en fazla 350 g hücreleri aşağı Spin ve pelet PBS içinde tekrar süspansiyon haline getirin. Hemasitometre kullanarak yeniden süspanse hücre sayımı. </li><li> 1x10 8 hücre / ml PBS içinde bir kap ile 5 ml steril bir polistiren tüp% 5, normal sıçan serumu (tüp başına 2 mL maksimum hacim) ile bir süspansiyon hazırlayın. </li><li> Ml için her hücre EasySep Negatif Seçim Fare B Hücre Zenginleştirme kokteyl 50 mcL ekleyin. Karışımı Pipet, tüpler kap ve 15 dakika boyunca buzdolabında yer. </li><li> Her hücre ml için 100 mcL EasySep Biotin Seçim Kokteyl ekleyin. Karışımı Pipet, tüpler kap ve 15 dakika boyunca buzdolabında yer. </li><li> EasySep Manyetik Nanopartiküller hücrelerin her ml için 100 mcL (nanopartiküller iyice karıştırılır ve çözüm homojen sağlamak) ekleyin. Karışımı Pipet, tüpler kap ve 5 dakika buzdolabına koyun. </li><li> PBS ve 5 dakika için bir EasySep mıknatıs yer 2.5ml Top çözüm. Mıknatıs ve tüp ters çevirin ve hızla, yeni bir polistiren tüp içine dökün. Olumsuz seçilen hücreleri manyetik tüp duvarları bağlı olacak gibi tüp sallamayın. </li><li> Saflık hücre süspansiyonu daha da artırmak için, tüp ek bir 5 dakika için cazibe merkezi haline yerleştirilemeyen ve yeni bir tüp dökülür olabilir. Bu genel hücre kurtarma azaltabilir. </li><li> B hücre saflık, B hücre yüzey belirteçleri akış sitometrik analizi ile tespit edilebilir. Zenginleştirme ardından, biz sürekli olarak&gt;% 95 B220 pozitif hücrelerin bir saflığa bakın. </li></ol> Adım: II KAKE hücre boyama <ol><li> Ml başına 1 x 10 6 hücre bir final konsantrasyonda albumin% 0.1 sığır serumu ile desteklenmiş sıcak PBS içinde izole hücrelerin tekrar </li><li> KAKE (steril dimetil sülfoksit seyreltilmiş) bir 5mM stok solüsyonu hazırlayın ve tüp hücrelerin her ml için 2μL ekleyin. 10μM son konsantrasyonu birincil B hücreleri boyama için en uygunudur. </li><li> 37 inkübe ° C de 10 dakika karanlıkta (not: KAKE ışığa duyarlı ve mümkün olduğunda karanlık tutulmalıdır). </li><li> Sığır buzağı serum eşit miktarda hücrelere ekleyerek 5 dakika buz üzerinde inkübe leke Quench. </li><li> Kültür ortamı ile tüpler g. (tarifi için adım III) tepesi ve 350 santrifüj hücreleri tarafından yıkayın Medyanın en az 5 kat daha hacimli eşdeğer, sığır buzağı serum viskozitesi karşı ve hücreler için bir pelet üretmek için eklenmesi gerektiğini unutmayın. </li><li> Hücrelerin kültür ortamında iki kez daha yıkayın. Hücrelerinin uyarılması için hazırız. </li></ol> Adım III: LPS ile hücre uyarımı <ol><li> % 10 fetal sığır serumu ve 50 mcM β-mercaptoethanol / RPMI 1640 Ürünü in vitro B hücre kültür ortamı hazırlayın. </li><li> 50μg/mL son bir konsantrasyonda LPS ekleyerek stimülasyon orta hazırlayın. Düz dipli her bir kuyunun 96-iyi doku kültürü plakası stimülasyon orta kısım 125 mcL. </li><li> 3.2 x 10 6 hücre / ml son bir konsantrasyonda LPS olmadan KAKE lekeli B hücreleri kültür ortamı hazırlayın. Stimülasyon orta ve hafifçe pipetleme karışım içeren kuyu hücre süspansiyonu 125 mcL ekleyin. </li><li> Her iyi şimdi 25 mcg / mL nihai LPS konsantrasyonu 4 x 10 5 hücreler içerir. Bu değerlerde değişiklikler istediğiniz gibi olsa da bu, hücre çoğalması ve sınıf değiştirme optimal düzeyde sağlar. </li><li> Hücreler 37 ° ° C&#39;de ve% 5 CO 2 72 96 saat. 48 saat sonra, büyük bir hızla çoğalan B hücreleri kümeleri, mikroskop altında açıkça görülebilir. </li></ol> Adım IV: sınıf anahtarlama Flow sitometrik analizi <ol><li> Sınıfı anahtarlama bakmak için hazır olduğunuzda, kendi kültür koşulları hücreleri çıkarmak ve 1 mL boyama tamponu (% 2 büyükbaş buzağı serumu ile PBS) iki kez yıkayın. </li><li> 350 g ve inkübasyon onları aşağı iplik hücreleri, pelet hücreler non-spesifik antikor boyama önlemek için% 5 normal fare serumu ve 15 dakika boyunca buz üzerinde FcBlock per106 hücrelerinin 1μg tampon boyama, 100μL te onları. Hücre engelleme buz üzerinde yapılmalıdır. </li><li> Hücrelerin yıkama olmadan, floresan etiketli anti-fare IgG3 antikor 10 6 hücre başına 1μg eklemek ve izin hücreler 30 dakika boyunca buz üzerinde leke. </li><li> 200-300 mcL boyama tampon santrifüj ve tekrar süspansiyon hücreler iki kez yıkayın. Hücreler flow sitometri ile değerlendirilmesi için hazırız. </li><li> KAKE 492 nm&#39;de optimal heyecan (argon-iyon lazer) ve 517 nm dalga boyunda yayar. IgG3 antikorunun eksitasyon ve emisyon spektrumu konjuge floresan boya bağlıdır. </li></ol> Temsilcisi Sonuçlar Manyetik zenginleştirme sonra, hücre süspansiyonu ayırt edilemeyen küçük dairesel hücreleri (Şekil 1A) homojen bir nüfus gibi görünmelidir. 48-72 saat sonra stimülasyon, genişlemiş hızla çoğalan hücreleri izole salkım (Şekil 1B) açıkça görülebilir. Apoptozis tabi olmayan hızla çoğalan hücrelerin büyük bir kısmı küçük ve granül görünecektir. KSS normal hücrelerin en çok 7 ölmeye başlamadan önce stimülasyon sonrasında 72 ve 96 saat arasında en yüksek seviyelerde tespit edilebilir. Bu aşamada, hücreleri, hücre bölünmeleri daha sonra kızı hücre popülasyonu görünen anahtarlamalı hücreleri ile bir dizi geçirmiş olmalı. LPS stimülasyon 96 saat sonra IgG3 KAKE seyreltme ve yüzey ekspresyonu bir temsilcisi akış sitometrik arsa Şekil 2&#39;de görülebilir. Tablo 1: geçiş kokteyller İzotip. Not: Uyarıcı konsantrasyonu değerleri yalnızca tavsiyelerdir. Titrasyonları gerekli olabilir. <table border="1"><tbody><tr><td> İstenen İzotip </td><td> Uyarım Kokteyl </td></tr><tr><td> IgG1and IgE </td><td> LPS (25μg/mL) ve IL-4 (10 ng / ml) </td></tr><tr><td> IgG1and IgE </td><td> Anti-CD40 (10μg/mL) ve IL-4 (10 ng / ml) </td></tr><tr><td> IgG2a </td><td> LPS (25μg/mL) ve IFN-γ (10 ng / ml) </td></tr><tr><td> IgG2b ve IgG3 </td><td> LPS (25μg/mL) </td></tr><tr><td> IgA </td><td> LPS (25μg/mL), TGF-β (2 ng / ml) ve IL-5 (1.5 ng / ml) </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="Şekil 1" /> Şekil 1 İzolasyon ve dalak B hücreleri LPS stimülasyon. (A) Manyetik LPS stimülasyon önce dalak B hücreleri zenginleştirilmiş. (B) 48 saat sonra 25 mg / ml LPS ile uyarılması. Proliferatif hücreler, kumlama ve apoptotik hücrelerin bir arka plan küme olarak görülür. <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="Şekil 2" /> Şekil 2, 96 saat sonra Silahların Yayılmasının Önlenmesi ve sınıf anahtarı rekombinasyon . (A) LPS için KAKE seyreltme, 96 saat sonra kültür hücreleri uyarılır. Her bağımsız zirve bağımsız bir hücre bölünmesi ile ilgili KAKE floresan bir seyreltme temsil eder. (B) IgG3 ifade, hücre bölünmesinin birkaç tur sonra bir IgG3 pozitif nüfusunun ortaya çıkması gösteriyor.

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

Yukarıda özetlenen protokol birincil fare B hücreleri sınıfı anahtarlama sırasında YARDIM ifade ve fonksiyonunu analiz etmek için standart bir tahlil sağlar. Bu protokol, diğer isotypes (Tablo 1 8, 9 özetlenmiştir) geçiş ikna etmek için uyarılması medya değişiklikler yapılabilir, ancak bakteriyel LPS, IgM gelen IgG3 geçiş ikna etmek için kullanır . Biz henüz bilinmeyen nedenlerden dolayı, fetal buzağı serumu sınıf in vitro gözlenen anahtarlama düze...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 Culture Medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>Lipopolysaccharides from Escherichia coli</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>L6529</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>011-000</td>
<td>&nbsp;</td>
<tr>
<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553141</td>
<td>&nbsp;</td>
<tr>
<td>Rat Anti-Mouse IgG3</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553401</td>
<td>&nbsp;</td>		</tbody>
		</table>
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induction and assessment of class switch recombination in purified murine b cells

Humoral bağışıklık, B hücreleri ve antikor salgılanmasını aracılığıyla tarafından yapılmaktadır bağışıklık sisteminin dalıdır. B hücre aktivasyonu üzerine, immünglobulin lokus bağlama kapasitesi ve salgılanan antikorlar effektör fonksiyonu değiştirmek için bir dizi genetik değişiklikler geçer. Bu süreç olduğu varsayılan IgM, IgG, IgA, veya IgE antikor izotip yerine sınıf anahtarı rekombinasyon (KSS) olarak bilinen bir genomik rekombinasyon olayı tarafından vurgulanır. Her izotip, böylece bağışıklık bakım KSS önemli hale farklı efektör fonksiyonlarına sahiptir. Immünglobulin lokus çeşitlendirilmesi enzim aktivasyonla indüklenen sitidinin deaminaz (AID) aracılığı ile. Bu süreci ayrıntılı olarak açıklayan bir şematik video online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). YARDIM faaliyet ve KSS yolağının genellikle B hücre fonksiyonu ve farelerde humoral bağışıklığın değerlendirilmesi incelenir. Bu raporda açıklanan protokol fare dalak ve sınıf diğer antikor isotypes (bkz. Tablo 1) IgG3 geçiş ikna etmek için bakteriyel lipopolisakkarit (LPS) ile sonraki stimülasyon B hücre izolasyonu bir yöntem sunuyor. Buna ek olarak, floresan hücre boyama boya Carboxyfluorescein succinimidyl ester (KAKE) uyarılan hücreler hücre bölünmesi, anahtarlama 1, 2 izotip için çok önemli bir süreç izlemek için kullanılır. YARDIM yönetmelik ve KSS oluştuğu mekanizması hala net değildir ve bu nedenle, in vitro sınıf anahtarı tayinlerde bu süreçlerin çeşitli fare modelleri test etmek için güvenilir bir yöntem sağlar. Bu testleri daha önce, knockout farelerde 3 kullanarak gen eksikliği bağlamında kullanılmaktadır . Ayrıca, in vitro B hücreleri anahtarlama, RNA demonte veya transgen ifade 4-6 gen ekspresyonu modüle viral transdüksiyon öncesinde olabilir . Deneyler bu tür veriler YARDIM aktivite, KSS reaksiyon çözünürlük ve fare antikor-aracılı bağışıklık anlayışımızı etkiledi.

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Antijen maruz kalma ardından, aktive B hücreleri alt popülasyonlar farklı efektör fonksiyonları ile antikor isotypes üretmek için sınıf anahtarı rekombinasyon (KSS) olarak bilinen bir süreç geçmesi. Bu raporda açıklanan protokol KSS uyarılan ve nasıl analiz edilebilir açıklıyor<em&gt; In vitro</em&gt; B hücre fonksiyonu eğitim amaçlı.

Sınıf Anahtarı Rekombinasyon murin B Hücreleri Saf İndüksiyon ve Değerlendirme

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

induction-assessment-class-switch-recombination-purified-murine-b

Research

JoVE Journal

Immunology and Infection

18.0K Görüntüleme.  University of Toronto. Antijen maruz kalma ardından, aktive B hücreleri alt popülasyonlar farklı efektör fonksiyonları ile antikor isotypes üretmek için sınıf anahtarı rekombinasyon (KSS) olarak bilinen bir süreç geçmesi. Bu raporda açıklanan protokol KSS uyarılan ve nasıl analiz edilebilir açıklıyor In vitro B hücre fonksiyonu eğitim amaçlı.

Video: Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

İmmünoloji ve Enfeksiyon

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Saf Hematopoetik kök / progenitör hücrelerin DNA mutasyonları belirlenmesi

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Doğru Algılama Fare B Lineage Hücreleri IgE-ifade

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

İnsan Mezenkimal Kök Hücre İmmünomodüler Properties (MKH) değerlendirilmesi

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Genom çapında modülasyon mikroRNA ve mRNA'ların B hücreleri indüklenen geçirmek sınıf--düğme DNA rekombinasyon ve Plazma hücre farklılaşması içinde analiz HDAC inhibitörü-aracılı

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

Vitro ve In Vivo değerlendirme T, B ve Myeloid hücrelerin süpresif etkinlik ve nakli alıcılardan gelen Humoral yanıt

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Yamultma akış kaynaklı geçiş antikorundan marjinal bölge B hücrelerinin in Vitro analizine

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.